Project Summary Laboratory animal models play an important role in the study of human diseases. Using appropriate animals is critical not only for basic research but also for the development of therapeutics and diagnostic tools. The rabbit is a classical experimental animal species. By adapting the programmable nucleases, such as CRISPR/Cas9, to the gene targeting platform, we recently achieved efficient gene knockout and knock-in in rabbits. It is realized that inducible and conditional gene targeting tools, such as safe harbor locus, Cre-loxP, and Tet, are still lacking which becomes a limiting factor for the broad applicability of this important large animal model system in biomedical research. We previously characterized the rabbit Rosa26 locus, demonstrated that it can be used as a safe harbor locus, and produce Rosa26-EGFP loxed rabbits. We have also identified another putative safe harbor locus, ortholog of Hipp11 (H11), on Chromosome 21 in the rabbit genome. In the present project we propose to: (i) develop conditional knockout (cKO) rabbits; and (ii) develop inducible knockout (iKO) rabbits. To achieve the cKO and iKO goals, we will utilize the rabbit H11 (rbH11) locus as the second safe harbor locus for knock-in work, and to develop Cre-loxP, CreER-loxP and Tet on/off tools. Furthermore, we propose to develop Cas9 based cKO/iKO tools. Through the propose work, we will generate versatile tools to enable conditional and inducible gene targeting tools in rabbits. These tools will realize spatial and temporal control of gene expressions in this model species.